The present invention relates to novel oligonucleotides which contain modified bases and which possess valuable physical, biological and pharmacological properties, to a process for their preparation and to their use as inhibitors of gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex-forming oligonucleotides), as probes for detecting nucleic acids, as aids in molecular biology and as pharmaceuticals or diagnostic agents.
Numerous chemical modifications of oligonucleotides are known from the literature. These modifications can affect the sugar-phosphate skeleton or the nucleotide bases. Reviews of the state of the art are provided, for example, by Uhlmann and Peyman, Chem. Rev. 1990, 90, 543 and Milligan et al., J. Med. Chem. 1993, 36, 1923.
As a rule, it is necessary to modify oligonucleotides chemically since unmodified oligonucleotides are very rapidly degraded by nucleolytic activities both in cells and in the cell culture medium. Stabilization against nucleolytic degradation can be achieved by replacing the sugar-phosphate backbone or by modifying the phosphate bridge, the sugar moiety or the nucleotide base [Milligan et al., above and Uhlmann and Peyman, above].
In addition to modifications which lead to oligonucleotides which possess increased stability towards nucleolytic degradation, those modifications are also of interest which alter the hybridization behavior of the modified oligonucleotides such that the latter are able, for example, to form more stable hybridization complexes (duplexes) with intracellular nucleic acids (so-called target nucleic acids). It is possible to alter the hybridization properties of oligonucleotides by, for example, modifying the bases. The altered hybridization properties of oligonucleotides which have been modified in this way can be recognized, for example, from the melting temperatures (Tm values) of the duplexes, which temperatures are different from those obtained with the unmodified oligonucleotides.
Thus, oligonucleotides which contain 5-bromouracil, for example, form more stable hybridization complexes with the complementary nucleic acids than do oligonucleotides which contain the corresponding, unsubstituted bases (uracil) [G. D. Fasman, CRC Handbook of Biochemistry and Molecular Biology, 3rd edition, 1975, Nucleic Acids, Vol. I, 58-585].
In addition, PCT Application WO 93/10820 discloses oligonucleotides which contain modified uracil or cytosine bases and which are able to form duplex or triplex structures with the target nucleic acids which are more stable than those formed by the unmodified oligonucleotides. Oligonucleotides which contain the base analog 2-aminoadenine have also been reported to exhibit improved hybridization properties [Chollet et al., (1988), Nucleic Acid Research, 16, 305-317]. German Patent Application P4415370.8 discloses that incorporating 8-azapurine bases into oligonucleotides increases the stability of the corresponding hybridization complexes with the target nucleic acids. In addition WO 93/09127 discloses oligonucleotides which contain substituted or unsubstituted 7-deazapurine bases and which, as a result, are more readily able to form triplex structures with the target molecules (double-stranded DNA). Oligonucleotides which contain 7-deazapurine bases which are substituted in the 7 position are also disclosed in WO 94/22892 and WO 94/24144.
However, it is not possible to predict the base modifications which will lead to an increase in duplex stability. Thus, numerous examples are known of base modifications which diminish duplex stability. Thus, PCT Application WO 92/002258 describes pyrimidine-modified oligonucleotides which exhibit decreased binding affinity for the target nucleic acids. Methyl or bromine substituents which are introduced at the 8 position of the purine ring also decrease the stability of the corresponding duplexes [E. N. Kanaya et al., Biochemistry (1987) 26 7159, and Biochemistry, 1984, 23, 4219]. Oligonucleotides which contain 7-deazaadenine form significantly fewer stable duplexes with complementary oligonucleotides than do oligonucleotides which contain adenine [Seela et al., Nucleic Acid Research (1982) 10, 1389].
The object of the present invention is, therefore, to make available novel oligonucleotides which possess advantageous properties.
It has now been found, surprisingly, that oligonucleotides which possess at least one substituted 7-deazapurine base form hybridization complexes with the target nucleic acids which are significantly more stable than those formed by comparable oligonucleotides which possess unsubstituted 7-deazapurine bases.
The invention consequently relates to oligonucleotides of the formula I 
and the physiologically tolerated salts thereof, in which
B are, independently of each other, a base which is customary in nucleotide chemistry, and at least one B is a base of the formula II 
xe2x80x83in which
R15 and R16 are, independently of each other,
1. hydrogen,
2. halogen,
3. (C1-C10)-alkyl,
4. (C2-C13)-alkenyl,
5. (C2-C10)-alkynyl,
6. NO2,
7. NH2,
8. cyano,
9. xe2x80x94Sxe2x80x94(C1-C6)-alkyl,
10. (C1-C6)-alkoxy,
11. (C6-C20)-aryloxy,
12. SiH3,
13. 
14. a radical as described under 3., 4. or 5. which is substituted by one or more radicals from the group SH, Sxe2x80x94(C1-C6)-alkyl, (C1-C6)-alkoxy, OH, xe2x80x94NR(c)R(d), xe2x80x94COxe2x80x94R(b), xe2x80x94NHxe2x80x94COxe2x80x94NR(c)R(d), xe2x80x94NR(c)R(g), xe2x80x94NR(e)R(f) or xe2x80x94NR(e)R(g), or by a polyalkyleneglycol radical of the formula xe2x80x94[Oxe2x80x94(CH2)r]sxe2x80x94NR(c)R(d), where r and s are, independently of each other, an integer between 1 and 18, preferably between 1 and 6, with it being possible for functional groups such as OH, SH, xe2x80x94COxe2x80x94R(b), xe2x80x94NHxe2x80x94COxe2x80x94NR(c)R(d), xe2x80x94NR(c)R(d), xe2x80x94NR(e)R(f), xe2x80x94NR(e)R(g) or xe2x80x94NR(c)R(g) additionally to be linked to one or more groups, where appropriate via a further linker, which favor intracellular uptake or serve for labeling a DNA or RNA probe, or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, or
15. a radical as defined under 3., 4. or 5. in which from one to all the H atoms are substituted by halogen, preferably fluorine;
R(a) is OH, (C1-C6)-alkoxy, (C6-C20)-aryloxy, NH2 or NHxe2x80x94T, where T is an alkylcarboxyl group or alkylamino group which is linked to one or more groups, where appropriate via a further linker, which favor intracellular uptake or serve for labeling a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, R(b) is hydroxyl, (C1-C6)-alkoxy or xe2x80x94NR(c)R(d), R(c) and R(d) are, independently of each other, H or (C1-C6)-alkyl which is unsubstituted or substituted by xe2x80x94NR(e)R(f) or xe2x80x94NR(e)R(g), R(e) and R(f) are, independently of each other, H or (C1-C6)-alkyl, R(g) is (C1-C6)-alkyl-COOH; with the proviso that R15 and R16 cannot each simultaneously be hydrogen, NO2, NH2, cyano or SiH3;
E and F are, independently of each other, H, OH or NH2,
R1 is hydrogen, C1-C18-alkyl, C2-C18-alkenyl, C2-C18-alkynyl, C2-C18-alkylcarbonyl, C3-C19-alkenylcarbonyl, C3-C19-alkynylcarbonyl, (C6-C14)-aryl (C1-C8)-alkyl, a protective group which is customary in nucleotide chemistry, or a radical of the formula IIIa 
R1a is hydrogen, C1-C8-alkyl, C2-C18-alkenyl, C2-C18-alkynyl, C2-C18-alkylcarbonyl, C3-C19-alkenylcarbonyl, C3-C19-alkynylcarbonyl, (C6-C14)-aryl-(C1-C8)-alkyl, or a radical of the formula IIIb 
R2 is hydrogen, hydroxyl, C1-C18-alkoxy , C1-C6-alkenyloxy, in particular allyloxy, halogen, in particular F, azido or NH2;
a is oxy, sulfanediyl or methylene;
n is an integer xe2x89xa71;
W is oxo, thioxo or selenoxo;
V is oxy, sulfanediyl or imino;
Y is oxy, sulfanediyl, imino or methylene;
Yxe2x80x2 is oxy, sulfanediyl, imino, (CH2)m or V(CH2)m, in which
m is an integer from 1 to 18;
X is hydroxyl or mercapto;
U is hydroxyl, mercapto, SeH, C1-C18-alkoxy, C1-C18-alkyl, C6-C20-aryl, (C6-C14)aryl-(C1-C8)-alkyl, NHR3, NR3R4 or a radical of the formula IV
(OCH2CH2)pO(CH2)qCH2R5xe2x80x83xe2x80x83(IV)
xe2x80x83in which
R3 is C1-C18-alkyl, C6-C20-aryl, (C6-C14)-aryl-(C1-C8)-alkyl, xe2x80x94(CH2)cxe2x80x94[NH(CH2)c]dxe2x80x94NR6R6, in which c is an integer from 2 to 6 and d is an integer from 0 to 6, and R6 is, independently of each other, hydrogen or C1-C6-alkyl or C1-C4-alkoxy-C1-C6-alkyl;
R4 is C1-C18-alkyl, C6-C20-aryl or (C6-C10)-aryl-(C1-C8)-alkyl, or, in the case of NR3R4, is, together with R3 and the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which can additionally contain a further heteroatom from the series O, S and N,
p is an integer from 1 to 100,
q is an integer from 0 to 22,
R5 is hydrogen or a functional group such as, for example, hydroxyl, amino, C1-C18-alkylamino, COOH, CONH2, COO(C1-C4)-alkyl or halogen;
Z and Zxe2x80x2 are, independently of each other, hydroxyl, mercapto, SeH, C1-C22-alkoxy, xe2x80x94Oxe2x80x94(CH2)bxe2x80x94NR6R7, in which b is an integer from 1 to 6, and R7 is C1-C6-alkyl or R6 and R7, together with the nitrogen atom carrying them, form a 3-6-membered ring, C1-C18-alkyl, C6-C20-aryl, (C6-C14)-aryl-(C1-C8)-alkyl, (C6-C14)-aryl-(C1-C8)-alkoxy, where is also heteroaryl and aryl is optionally substituted by 1, 2 or 3 identical or different radicals from the group carboxyl, amino, nitro, C1-C4-alkylamino, C1-C6-alkoxy, hydroxyl, halogen and cyano, C1-C18-alkylmercapto, NHR3, NR3R4, a radical of the formula IV or a group which favors intracellular uptake or serves for labeling a DNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attacks the latter while binding, cross-linking or cleaving, and
the curved bracket indicates that R2 and the adjacent phosphoryl or xe2x80x94Yxe2x80x2xe2x80x94R1a radical can either be located in the 2xe2x80x2 and 3xe2x80x2 positions or else, conversely, in the 3xe2x80x2 and 2xe2x80x2 position, with it being possible for each nucleotide to be present in its D or L configuration, and for the base B to be located in the xcex1 or xcex2 position.
Bases which are customary in nucleotide chemistry are to be understood to mean, for example, natural bases, such as adenine, cytosine, thymine, guanine, uracil or hypoxanthine, or unnatural bases, such as, for example, purine, 8-azapurine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine, N4,N4-ethanocytosine, N6,N6-ethano-2,6-diaminopurine, pseudoisocytosine, 5-methylcytosine, 5-fluorouracil, 5-(C3-C6)-alkynyluracil, 5-(C3-C6)-alkynylcytosine, or their prodrug forms.
Oligonucleotides of the formula I are preferred which possess at least one 7-deazaguanine base (E is NH2 and F is OH) or 7-deazaadenine base (E is H and F is NH2) which is substituted at the 7 position.
Oligonucleotides of the formula I are particularly preferred which possess at least one 7-deazaadenine base which is substituted at the 7 position and, where appropriate, one or more 7-deazaguanine bases which are substituted at the 7 position, in addition.
Oligonucleotides of the formula I which possess at least one 7-deazapurine base which is substituted at the 7 and 8 positions (=disubstituted 7-deazapurine bases) represent a further preferred embodiment of the present invention. Oligonucleotides of the formula I having disubstituted 7-deazapurine bases are preferred in which the disubstituted 7-deazapurine bases carry a substituent at the 8 position which is defined under R16 2., 3., 4., 5., 14. and 15. A halogen, for example fluorine, is particularly preferred at the 8 position. The substituents defined under R15 3., 4., 5., 14. and 15., in particular hexynyl, are preferred substituents at the 7 position. Oligonucleotides of the formula I are also preferred in which
R15 is 1. NO2,
2. NH2,
3. xe2x80x94Sxe2x80x94(C1-C6)-alkyl,
4. (C1-C6)-alkoxy,
5. (C6-C20)-aryloxy,
6. SiH3,
7. 
8. (C1-C10)-alkyl, (C2-C10)-alkenyl or (C2-C10)-alkynyl which is substituted by one or more radicals from the group SH, Sxe2x80x94(C1-C6)-alkyl, (C1-C6)-alkoxy, OH, xe2x80x94NR(c)R(d), xe2x80x94COxe2x80x94R(b), xe2x80x94NHxe2x80x94COxe2x80x94NR(c)R(d), xe2x80x94NR(c)R(g), xe2x80x94NR(e)R(f) or xe2x80x94NR(e)R(g), or by a polyalkylene glycol radical of the formula xe2x80x94[Oxe2x80x94(CH2)r]sxe2x80x94NR(c)R(d), where r and s are, independently of each other, an integer between 1 and 18, preferably 1 and 6, with it being possible for functional groups such as OH, SH, xe2x80x94COxe2x80x94R(b), xe2x80x94NHxe2x80x94COxe2x80x94NR(c)R(d), xe2x80x94NR(c)R(d), xe2x80x94NR(e)R(f), xe2x80x94NR(e)R(g) or xe2x80x94NR(c)R(g) additionally to be linked to one or more groups, where appropriate via a further linker, which favor intracellular uptake or serve for labeling a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, or
9. (C1-C10)-alkyl, (C2-C10)-alkenyl or (C2-C10)-alkynyl in which from one to all the H atoms are substituted by halogen, preferably fluorine; and
R16 is hydrogen.
If only the 7 position of the 7-deazapurine bases is substituted (R16=H), C1-C10-alkyl, C2-C10-alkenyl or C2-C10-alkynyl radicals, in which from one to all the H atoms are substituted by halogen, preferably fluorine, are then particularly preferred for the 7 position.
Generally, 7-deazapurine-containing oligonucleotides are preferred in which the 7-deazapurine bases bear electron-attracting substituents at the 7 position and/or 8 position.
Oligonucleotides of the formula I are also preferred in which the base is located in the xcex2 position on the sugar, the nucleotides are in the D configuration, R2 is located in the 2xe2x80x2 position and a is oxy.
The 7 position of the azapurine ring system is to be understood to mean the position at which the substituent R15 is located. Correspondingly, the substituent R16 is located at the 8 position.
When attaching to complementary nucleic acids (target nucleic acids), the novel oligonucleotides exhibit a binding affinity which is superior to that exhibited by the natural oligonucleotides.
A further advantage of the novel oligonucleotides is that their stability towards acids and nucleases is increased as compared with that of oligonucleotides which contain natural purine bases.
It is advantageous if additional modifications, for example of the phosphate backbone, the ribose unit or the oligonucleotide ends, are introduced into these oligonucleotides when they are to be used therapeutically [J. S. Cohen, Topics in Molecular and Structural Biology 12 (1989) Macmillan Press, E. Uhlmann et al., above]. For example, modifications, which are known per se, of the sugar phosphate backbone result in the novel oligonucleotides becoming even more efficiently protected against nuclease attack, which is advantageous.
Compounds of the formula I are also preferred, therefore, in which V, Y, Yxe2x80x2 and W have the meaning of thioxo, selenoxo, oxy, oxo, sulfanediyl, imino or methylene, and U has the meaning of hydroxyl, mercapto or methyl. These compounds are very particularly preferred if R2 additionally is hydroxyl or hydrogen, in particular hydrogen.
Compounds of the formula I in which R1 and R1a are hydrogen also represent a preferred embodiment.
Compounds of the formula I are very particularly preferred in which R1 and/or R1a is hydrogen, R2 is hydroxyl or hydrogen, U is hydroxyl or mercapto, and V, Y, Yxe2x80x2 and W have the meaning of thioxo, oxy, oxo or hydroxyl.
Protective groups which are customary in nucleotide chemistry are to be understood to mean, for example, amino protective groups, hydroxyl protective groups or other protective groups as described in [E. Sonveaux, 1986, Bioorganic Chemistry, 14, 274-325 or S. L. Beaucage et al., 1992, Tetrahedron, 48, 2223-2311].
Alkyl, alkenyl and alkynyl may be straight-chain or branched. The same also applies, in a corresponding manner, to radicals which are derived from them, such as alkanoyl or alkoxy. (C1-C10)-Alkyl is, in particular, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl and heptyl. Examples of halogenated (C1-C10)-alkyls are CHF2, CF3, CH2F, CF3xe2x80x94CH2xe2x80x94CH2xe2x80x94, CF3xe2x80x94CF2xe2x80x94CH2, CF3(CF2)6xe2x80x94CH2, 
(C2-C10)-Alkenyl is, for example, vinyl (xe2x80x94CHxe2x95x90CH2), 1-propenyl (xe2x80x94CHxe2x95x90CHxe2x80x94CH3), 2-methyl-1-propenyl (xe2x80x94CHxe2x95x90C(CH3)xe2x80x94CH3), 1-butenyl (xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x80x94CH3); 1-pentenyl, 1-hexenyl, 1-heptenyl and 1-octenyl. Examples of halogenated (C2-C10)-alkenyls are xe2x80x94CHxe2x95x90CF2, xe2x80x94CHxe2x95x90CHxe2x80x94CF3 and xe2x80x94CFxe2x95x90CFxe2x80x94CF3.
(C2-C10)-Alkynyl is, for example, ethynyl (xe2x80x94Cxe2x89xa1CH), 1-propynyl (xe2x80x94Cxe2x89xa1Cxe2x80x94CH3), 1-butynyl (xe2x80x94Cxe2x89xa1Cxe2x80x94CH2xe2x80x94CH3), 3-methyl-butynyl (xe2x80x94Cxe2x89xa1Cxe2x80x94CH(CH3)xe2x80x94CH3), 3,3-dimethyl-butynyl (xe2x80x94Cxe2x89xa1Cxe2x80x94C(CH3)3), 1-pentynyl, 1,3-pentadiynyl (xe2x80x94Cxe2x89xa1Cxe2x80x94Cxe2x89xa1Cxe2x80x94CH3), 1-hexynyl and 1-heptynyl. Examples of halogenated (C2-C10)-alkynyls are xe2x80x94Cxe2x89xa1Cxe2x80x94CH2F, xe2x80x94Cxe2x89xa1Cxe2x80x94CF3, xe2x80x94Cxe2x89xa1Cxe2x80x94(CH2)3xe2x80x94CF3 and xe2x80x94Cxe2x89xa1Cxe2x80x94(CF2)3xe2x80x94CF3,
Cycloalkyl is also understood to mean alkyl-substituted rings.
(C6-C20)-Aryl is, for example, phenyl, naphthyl or biphenylyl, preferably phenyl.
Halogen is to be understood to mean iodine, bromine, chlorine or fluorine.
Heteroaryl is understood to mean, in particular, radicals which are derived from phenyl or naphthyl in which one or more CH groups are replaced by N and/or in which at least two adjacent CH groups are replaced (with the formation of a five-membered aromatic ring) by S, NH or O. In addition, one or both atoms of the condensation site of bicyclic radicals can be N atoms (as in indolizinyl). Heteroaryl is, in particular, furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, indazolyl, quinolyl, isoquinolyl, phthalazinyl, quinoxalinyl, quinazolinyl and cinnolinyl.
The morpholinyl radical and the imidazolidinyl radical may be mentioned as examples of NR3R4 groups in which R3 and R4, together with the nitrogen atom carrying them, form a 5- to 6-membered heterocyclic ring which additionally contains a further heteroatom.
Physiologically tolerated salts of compounds of the formula (I) are understood to mean both inorganic and organic salts, as described in Remington""s Pharmaceutical Sciences (17th edition, page 1418 (1985)).
Owing to their physical and chemical stability, and their solubility, sodium salts, potassium salts, calcium salts and ammonium salts, inter alia, are preferred for acidic groups.
The invention is not limited to xcex1- and xcex2-D- or L-ribofuranosides, xcex1- and xcex2-D- or L-deoxyribofuranosides and corresponding carbocyclic five-ring analogs, but also applies to oligonucleotide analogs which are assembled from other sugar building blocks, for example xylofuranose and arabinofuranose derivatives, ring-expanded and ring-contracted sugars, and acyclic and ring-bridged sugar derivatives or suitable sugar derivatives of a different kind. Furthermore, the invention is not limited to the derivatives of the phosphate radical which are listed by way of example in formula I, but also relates to the known dephospho derivatives.
Consequently, the novel oligonucleotides can result from modifying the natural structure in a variety of ways. Examples of such modifications, which are introduced by methods which are known per se, are:
a) Modifications of the Phosphate Bridge
The following may be mentioned by way of example: phosphorothioates, phosphorodithioates, methyl-phosphonates, phosphoroamidates, boranophosphates, methyl phosphates, ethyl phosphates and phenylphosphonates. Phosphorothioates, phosphorodithioates and methyl-phosphonates are preferred modifications of the phosphate bridge.
b) Replacement of the Phosphate Bridge
The following may be mentioned by way of example: replacement with acetamide, formacetal, 3xe2x80x2-thioformacetal, methylhydroxylamine, oxime, methylenedimethylhydrazo, dimethylenesulfone and silyl groups. Replacement with acetamide, formacetals and 3xe2x80x2-thioformacetals is preferred.
c) Modifications of the Sugar
The following may be mentioned by way of example: xcex1-anomeric sugars, 2xe2x80x2-O-methylribose, 2xe2x80x2-O-butylribose, 2xe2x80x2-O-allylribose, 2xe2x80x2-fluoro-2xe2x80x2-deoxyribose, 2xe2x80x2-amino-2xe2x80x2-deoxyribose, xcex1-arabinofuranose and carbocyclic sugar analogs. The preferred modification is that due to 2xe2x80x2-O-methylribose and 2xe2x80x2-O-n-butylribose.
d) Modifications of the Sugar and of the Phosphate Bridge
Those which may be mentioned by way of example are the peptide nucleic acids (PNA""s), in which the sugar/phosphate backbone is replaced by an aminoethylglycine backbone (see German Patent Application P4408531.1), and the carbamate-bridged morpholino oligomers. The PNA""s can also be linked to nucleic acids, as described in German Patent Application P4408528.1.
e) Other Modifications of the Bases, in Particular of the Pyrimidine Bases
The following may be mentioned by way of example: 5-propynyl-2xe2x80x2-deoxyuridine, 5-propynyl-2xe2x80x2-deoxycytidine, 5-hexynyl-2xe2x80x2-deoxyuridine, 5-hexynyl-2xe2x80x2-deoxycytidine, 5-fluoro-2xe2x80x2-deoxycytidine, 5-fluoro-2xe2x80x2-deoxyuridine, 5-hydroxymethyl-2xe2x80x2-deoxyuridine, 5-methyl-2xe2x80x2-deoxycytidine and 5-bromo-2xe2x80x2-deoxycytidine. 5-Propynyl-2xe2x80x2-deoxyuridine, 5-hexynyl-2xe2x80x2-deoxyuridine, 5-hexynyl-2xe2x80x2-deoxycytidine and 5-propynyl-2xe2x80x2-deoxycytidine are preferred modifications.
f) 3xe2x80x2-3xe2x80x2 and 5xe2x80x2-5xe2x80x2 Inversions [e.g. M. Koga et al., J. Org. Chem. 56 (1991) 37571]
g) 5xe2x80x2Conjugates and 3xe2x80x2-Conjugates.
Examples of groups which favor intracellular uptake are different lipophilic radicals, such as xe2x80x94Oxe2x80x94(CH2)xxe2x80x94CH3, in which x is an integer from 6 to 18, xe2x80x94Oxe2x80x94(CH2)nxe2x80x94CHxe2x95x90CHxe2x80x94(CH2)mxe2x80x94CH3, in which n and m are, independently of each other, an integer from 6 to 12, xe2x80x94Oxe2x80x94(CH2CH2O)4xe2x80x94(CH2)9xe2x80x94CH3, xe2x80x94Oxe2x80x94(CH2CH2O)8xe2x80x94(CH2)13xe2x80x94CH3 and xe2x80x94Oxe2x80x94(CH2CH2O)7xe2x80x94(CH2)15xe2x80x94CH3, and also steroid radicals, such as cholesteryl, or vitamin radicals, such as vitamin E, vitamin A or vitamin D, and other conjugates which exploit natural carrier systems, such as bile acid, folic acid, 2-(N-alkyl, N-alkoxy)-aminoanthraquinone and conjugates of mannose and peptides of the corresponding receptors which lead to receptor-mediated endocytosis of the oligonucleotides, such as EGF (epidermal growth factor), bradykinin and PDGF (platelet derived growth factor). Labeling groups are to be understood to mean fluorescent groups, for example of dansyl (=N-dimethyl-1-aminonaphthyl-5-sulfonyl) derivatives, fluorescein derivatives or coumarin derivatives, or chemiluminescent groups, for example of acridine derivatives, and also the digoxygenin system, which is detectable by means of ELISA, the biotin group, which is detectable by means of the biotin/avidin system, or else linker arms having functional groups which permit subsequent derivatization with detectable reporter groups, for example an amino-alkyl linker which is converted into the chemiluminescence probe using an acridinium active ester. Other suitable linkers are known to a person skilled in the art from the published patent applications EP 251786 and WO 93/09217.
h) Conjugation by way of the 7 Position and/or the 8 Position on the 7-Deazapurine
Groups which serve to label a DNA or RNA probe or which favor intracellular uptake can also be conjugated by way of the 7 position and/or 8 position of the 7-deazapurine. 7-Deazapurine nucleosides to which biotin or iminobiotin radicals are conjugated by way of the 7 position of the 7-deazapurine, via a special connecting group, have been disclosed by EP 63 879.
Labeling groups for a DNA or RNA probe are to be understood to mean fluorescent groups, for example of dansyl (=N-dimethyl-1-aminonaphthyl-5-sulfonyl) derivatives, fluorescein derivatives or coumarin derivatives, or chemiluminescent groups, for example of acridine derivatives, and also the digoxygenin system, which is detectable by means of ELISA, or the biotin group, which is detectable by means of the biotin/avidin system, and also the intercalators and chemically active groups which have already been listed under g) (see, also, Beaucage et al., Tetrah. (1993) Vol. 49, No. 10, 1925-1963). Examples of groups which favor intracellular uptake are steroid radicals, such as cholesteryl, or vitamin radicals such as vitamin E, vitamin A or vitamin D, and other conjugates which exploit natural carrier systems, such as bile acid, folic acid, 2-(N-alkyl, N-alkoxy)-aminoanthraquinone and conjugates of mannose and peptides of the corresponding receptors which lead to receptor-mediated endocytosis of the oligonucleotides, such as EGF (epidermal growth factor), bradykinin and PDGF (platelet derived growth factor).
In a general manner, the described groups can be introduced either at the level of the oligonucleotides (for example by way of SH groups) or at the level of the monomers (phosphonates, phosphoamidites or triphosphates). In the case of the monomers, in particular in the case of the triphosphates, it is advantageous to leave the side chains, into which a reporter group or an intercalator group is to be introduced, in the protected state, and only to eliminate the side-chain protective groups, and to react with an optionally activated derivative of the corresponding reporter group or intercalator group, after the phosphorylation.
Typical labeling groups are: 
Oligonucleotide analogs which bind to nucleic acids or intercalate with them and/or cleave or cross-link them, contain, for example, acridine, psoralene, phenanthridine, naphthoquinone, daunomycin or chloroethylaminoaryl conjugates. Typical intercalating and cross-linking radicals are: 
Fluorescein derivative
R=H or amino protective group 
Biotin conjugate (=xe2x80x9cBiotinxe2x80x9d for R=Fmoc) 
Acridine derivative x=2-12, preferably 4 
x=2-12, preferably 4 
Trimethylpsoralene conjugate (=xe2x80x9cPsoralenexe2x80x9d for X=0) 
Phenanthroline conjugate 
Psoralene conjugate 
Naphthoquinone conjugate 
Daunomycin derivative 
x=1-18, X=alkyl, halogen, NO2, 
x=1-18, X=alkyl, halogen, NO2, 
The invention furthermore relates to compounds of the formula V 
in which
V is oxy, sulfanediyl or imino;
Yb is oxy, sulfanediyl, imino or methylene;
a is oxy, sulfanediyl or methylene;
R2b is hydrogen, OR12, C1-C18-alkoxy, C1-C6-alkenyloxy, in particular allyloxy, halogen, azido or NR10R11;
R1 is a protective group which is customary in nucleotide chemistry;
R1b is a succinyl radical or other conventional linker for linking the oligonucleotide containing this group to a solid support e.g., an amino-functionalized or methylamino-functionalized support, by way of an amide or methylimide bond, or the like, or is a radical of the formula IIIc or IIId 
xe2x80x83in which
U is (C1-C18)-alkoxy, (C1-C18)-alkyl, (C6-C20)-aryl, (C6-C14)-aryl-(C1-C8)-alkyl, Oxe2x80x94R7, Sxe2x80x94R7 or a radical of the formula IV
(OCH2 CH2)pO(CH2)qCH2R5xe2x80x83xe2x80x83(IV)
in which R5 is H;
Q is a radical xe2x80x94NR8R9,
R7 is xe2x80x94(CH2)2xe2x80x94CN;
R8 and R9 are identical or different and are C1-C6-alkyl, in particular isopropyl or ethyl, or, together with the nitrogen atom carrying them, are a 5-9-membered heterocyclic ring which can additionally contain a further hetero atom from the series O, S and N, in particular 
Exe2x80x2 and Fxe2x80x2 are, independently of each other, H, OR12 or N10R11,
R10 and R11 are identical or different and are hydrogen or an amino protective group which is customary in nucleotide chemistry, or R10 and R11 together form an amino protective group which is customary in nucleotide chemistry,
R12 is hydrogen or a hydroxyl protective group which is customary in nucleotide chemistry, such as, for example, t-butyldimethyl-silyl, dimethoxytriphenylmethyl (DMT), triisopropyl-silyl, o-nitro-benzyl, p-nitro-benzyl, iBu, 2-fluorophenyl-4-methoxypiperidin-4-yl (FPMP), or methyl,
R15 and R16 are, independently of each other,
1. hydrogen,
2. halogen,
3. (C1-C10)-alkyl,
4. (C2-C10)-alkenyl,
5. (C2-C10)-alkynyl,
6. NO2,
7. NH2,
8. cyano,
9. xe2x80x94Sxe2x80x94(C1-C6)-alkyl,
10. (C1-C6)-alkoxy,
11. (C6-C20)-aryloxy,
12. SiH3,
13. 
14. a radical as defined under 3., 4. or 5. which is substituted by one or more radicals from the group SH, Sxe2x80x94(C1-C6)-alkyl, (C1-C6)-alkoxy, OH, xe2x80x94NR(c)R(d), xe2x80x94COxe2x80x94R(b), xe2x80x94NHxe2x80x94COxe2x80x94NR(c)R(d), xe2x80x94NR(c)R(g), xe2x80x94NR(e)R(f) or xe2x80x94NR(e)R(g), or by a polyalkyleneglycol radical of the formula xe2x80x94[Oxe2x80x94(CH2)r]sxe2x80x94NR(c)R(d), where r and s are, independently of each other, an integer between 1 and 18, preferably 1 and 6, with it being possible for functional groups such as OH, SH, xe2x80x94COxe2x80x94R(b), xe2x80x94NHxe2x80x94COxe2x80x94NR(c)R(d), xe2x80x94NR(c)R(d), xe2x80x94NR(e)R(f), xe2x80x94NR(e)R(g) or xe2x80x94NR(c)R(g) to carry a protective group which is customary in nucleotide chemistry or to be linked, where appropriate via a further linker, to one or more groups which favor intracellular uptake or serve as labeling for a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, or
15. a radical as defined under 3., 4. or 5. in which from one to all the H atoms are substituted by halogen, preferably fluorine.
R(a) is OH, (C1-C6)-alkoxy, (C6-C20)-aryloxy, NH2 or NHxe2x80x94T, where T is an alkylcarboxyl group or alkylamino group which is linked, optionally via a further linker, to one or more groups which favor intracellular uptake, or serve as labeling for a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving,
R(b) is hydroxyl, (C1-C6)-alkoxy or xe2x80x94NR(c)R(d),
R(c) and R(d) are, independently of each other, H or (C1-C6)-alkyl which is unsubstituted or substituted by xe2x80x94NR(e)R(f) or xe2x80x94NR(e)R(g),
R(e) and R(f) are, independently of each other, H or (C1-C6)-alkyl,
R(g) is (C1-C6)-alkyl-COOH, with the proviso that R15 and R16 cannot each simultaneously be hydrogen, NO2, NH2, cyano or SiH3, with functional groups such as OH, NH2 or COOH being protected, where appropriate, with a protective group which is customary in nucleotide chemistry, and the curved bracket indicating that R2b and the adjacent xe2x80x94Ybxe2x80x94R1b radical can be located in the 2xe2x80x2 and 3xe2x80x2 positions or else, conversely, in the 3xe2x80x2 and 2xe2x80x2 positions.
A preferred embodiment is represented by compounds of the formula (V) in which V, Yb and a are oxy, R2b is hydrogen or OR12, in particular hydrogen, and R1b is a radical of the formula (IIIc) or (IIId), with U being an Oxe2x80x94(CH2)2xe2x80x94CN, and R8 and R9 being identical or different and being isopropyl or ethyl, or, together with the N atom carrying them, being an aliphatic heterocycle, preferably pyrrolidino. These compounds are very particularly preferred if, in addition, the base is located in the a position on the sugar and R2b is located in the 2xe2x80x2 position. Compounds of formula (V) are also preferred in which E is NR10R11 and F is H, and, quite generally, those compounds of the formula (V) are preferred which can be employed for preparing preferred oligonucleotides of the formula I.
Examples of preferred amino protective groups are acyl or amidine protective groups.
The radical of the formula (IIId) which is customarily present as a salt is to be understood to mean inorganic or organic salts, for example alkali metal, alkaline earth metal or ammonium salts, which are described, for example, in Remington""s Pharmaceutical. Sciences (17th edition, page 1418 (1985)). Triethylammonium and pyridinium salts may be mentioned by way of example. However, the invention also embraces compounds of the formula (V) in which the radical of the formula (IIId) is present as a free acid.
The compounds of the formula V may be employed as structural components for preparing the novel oligonucleotides of the formula I.
EP 251 786 discloses 7-deazapurine nucleotides, and their monophosphates, diphosphates or triphosphates, which possess an alkynylamino group at the 7-purine position. The alkynylamino group serves as a linker by way of which fluorescent labeling molecules can be coupled to the nucleotide. The dideoxynucleotides which have been provided with a fluorescence label can then be used as chain terminator molecules for dideoxy sequencing in accordance with Sanger and detected directly by means of fluorescence spectroscopy. U.S. Pat. No. 5,241,060 discloses 7-deazapurine nucleotides which carry a detectable radical on the 7-deazapurine.
The invention also relates to compounds of the formula VI 
in which, independently of each other,
Uxe2x80x2=Uxe2x80x3=Uxe2x80x2xe2x80x3 is hydroxyl or mercapto, and Uxe2x80x2 can additionally be BH3,
e and f are 0 or 1;
R13 is hydrogen, OH, C1-C18-alkoxy, or C1-C6-alkenyloxy, in particular allyloxy;
E and F are, independently of each other, H, OH or NH2; and
R15 and R16 are, independently of each other,
1. hydrogen,
2. halogen,
3. (C1-C10)-alkyl,
4. (C2-C10)-alkenyl,
5. (C2-C10)-alkynyl,
6. NO2,
7. NH2,
8. cyano,
9. xe2x80x94Sxe2x80x94(C1-C6)-alkyl,
10. (C1-C6)-alkoxy,
11. (C6-C20)-aryloxy,
12. SiH3,
13. 
14. a radical as defined under 3., 4. or 5. which is substituted by one or more radicals from the group SH, Sxe2x80x94(C1-C6)-alkyl, (C1-C6)-alkoxy, OH, xe2x80x94NR(c)R(d), xe2x80x94COxe2x80x94R(b), xe2x80x94NHxe2x80x94COxe2x80x94NR(c)R(d), xe2x80x94NR(c)R(g), xe2x80x94NR(e)R(f) or xe2x80x94NR(e)R(g), or by a polyalkyleneglycol radical of the formula xe2x80x94[Oxe2x80x94(CH2)r]sxe2x80x94NR(c)R(d), where r and s are, independently of each other, an integer between 1 and 18, preferably 1 and 6, with it being possible for functional groups such as OH, SH, xe2x80x94COxe2x80x94R(b), xe2x80x94NHxe2x80x94COxe2x80x94NR(c)R(d), xe2x80x94NR(c)R(d), xe2x80x94NR(e)R(f), xe2x80x94NR(e)R(g) or xe2x80x94NR(c)R(g) additionally to be linked, where appropriate via a further linker, to one or more groups which favor intracellular uptake or serve as labeling for a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, or
15. a radical as defined under 3., 4. or 5. in which from one to all the H atoms are substituted by halogen, preferably fluorine.
R(a) is OH, (C1-C6)-alkoxy, (C6-C20)-aryloxy, NH2 or NHxe2x80x94T, with T representing an alkylcarboxyl or alkylamino group which is linked, where appropriate via a further linker, to one or more groups which favor intracellular uptake or serve as labeling for a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving,
R(b) is hydroxyl, (C1-C6)-alkoxy or xe2x80x94NR(c)R(d),
R(c) and R(d) are, independently of each other, H or (C1-C6)-alkyl which is unsubstituted or substituted by xe2x80x94NR(e)R(f) or xe2x80x94NR(e)R(g),
R(e) and R(f) are, independently of each other, H or (C1-C6)-alkyl,
R(g) is (C1-C6)-alkyl-COOH, with the proviso that R15 and R16 cannot each simultaneously be hydrogen, NO2, NH2, cyano or SiH3, with compounds of the formula VI being excepted in which R16 is H and R15 is (C2-C10)-alkynyl which is substituted by xe2x80x94NR(c)R(d) or xe2x80x94NR(e)R(f); and with the additional proviso that e and f are not 0 if E is OH or NH2 and F is OH, R16 is hydrogen and R15 is Br, Cl, F, cyano, (C1-C4)-alkyl, (C2-C4)-alkenyl or (C2-C4)-alkynyl.
The invention also embraces compounds of the formula VI which are provided, in a generally customary manner, with a radioactive label (for example, xcex1P atom is 32P; Uxe2x80x2 is 35S).
Compounds of the formula VI are preferred in which Uxe2x80x2 is hydroxyl or mercapto, Uxe2x80x3=Uxe2x80x2xe2x80x3 is hydroxyl, and e and/or f is 1. Compounds of the formula VI are particularly preferred when e and f are 1.
The compounds of the formula VI which are customarily present as a salt comprise inorganic or organic salts, for example alkali metal, alkaline earth metal or ammonium salts [Remington""s Pharmaceutical Sciences (17th edition, page 1418 (1985)]. Triethylammonium and pyridinium salts may be mentioned by way of example. The novel VI compounds also comprise those compounds in which the phosphate group is present as a free acid.
The novel compounds of the formula VI may be employed generally as aids in molecular biology, for example in PCR reactions (e=f=1, R13=OH) or for sequencing (e=f=1; R13=H or OH). In PCR-reactions compounds of the formula VI are preferred in which R16 is H and R15 is halogen. Amplification of longer nucleodide sequences is enhanced using the modified oligonucleotides.
The use of the novel 7-deazapurine nucleotides for sequencing nucleic acids is advantageous for several reasons. Thus, the band compression which can often be observed in GC-rich nucleotide regions in the Sanger sequencing method (dideoxy technique), and which hinders correct determination of the nucleotide sequence, is either eliminated or at least reduced. In addition, the double-stranded nucleic acids which are synthesized by DNA polymerases or RNA polymerases during the sequencing are stabilized by the incorporation of 7-, 8- or 7,8-substituted 7-deazapurine bases. It is consequently more advantageous to use substituted 7-deazapurine nucleotides than to use unsubstituted 7-deazaguanosine nucleotides, which are customarily employed in nucleic acid sequencing in order to eliminate band compressions in GC-rich DNA stretches (EP 212536). A further advantage of using substituted 7-deazapurine nucleotides in the sequencing is that fluorescent residues in the form of reporter groups, which make possible fluorescence-spectroscopic detection of the nucleic acid molecules which are synthesized during the sequencing reaction, can be introduced onto the substituents in a series of subsequent reactions.
In addition, the incorporation of self-fluorescent, substituted 7-deazapurine bases into oligonucleotides renders it possible to detect the latter directly by way of the self-fluorescence of the substituted 7-deazapurine bases. Thus, the 7-deazapurine bases, which in unsubstituted form are not fluorescent, become fluorescent, for example, when an alkynyl group, for example hexynyl, is introduced at the 7 position. The self-fluorescence of these compounds can be measured at 350 nm (emission) following excitation with light of 280 nm wavelength.
The compounds of the formula VI can be prepared by proceeding from the corresponding substituted 7-deazapurine nucleosides and using well known methods. The compounds of the formula VI can preferably be prepared by an abbreviated one-pot method due to Ludwig, in the presence of 1,8-bis(dimethylamino)naphthalene and trimethyl phosphate [J. Ludwig et al., (1981) Acta Biochem. Biophys. Sci. Hung., 16, 131].
The invention also relates to compounds of the formula VII 
in which
E and F are, independently of each other, H, OH or NH2, and OH and NH2 are, where appropriate, protected by a protective group which is customary in nucleotide chemistry;
R15 and R16 are, independently of each other, hydrogen, (C1-C10)-alkyl, (C2-C10)-alkenyl, (C2-C10)-alkynyl, I, Cl, Br, F, cyano, or (C1-C10)-alkyl, (C2-C10)-alkenyl or (C2-C10)-alkynyl in which from one to all the H atoms are substituted by halogen, preferably fluorine, with it not being possible for R15 and R16 to be simultaneously hydrogen and cyano, and with the further proviso that R15 is not I if R16 is hydrogen, E is NH2 and F is OH,
R14 are, independently of each other, H or a protective group which is customary in nucleotide chemistry.
The invention also embraces all the tautomeric forms of the compounds of the formulae I, V, VI and VII, and, in particular, all the tautomeric forms of the 7-deazapurine bases of the formula II.
In a quite general manner, those compounds of the formulae V, VI and VII are also preferred which can be used as starting compounds or intermediates for the preparation of preferred oligonucleotides of the formula I.
The invention furthermore relates to a process for preparing the novel oligonucleotides of the formula I. The standard conditions which are customary in the chemical synthesis of oligonucleotides can be applied for preparing the novel oligonucleotides containing substituted 7-deazapurine.
The novel oligonucleotides of the formula I are prepared in solution or, preferably, on a solid phase, where appropriate using an automatic synthesis device. The oligomers of the formula I can be assembled stepwise by successively condensing a mononucleotide, which in each case possesses a nucleotide base, onto an appropriately derivatized support or onto a growing oligomer chain. Alternatively, the oligonucleotides of the formula I can be assembled by joining dinucleotides or trinucleotides together [S. Beaucage et al., Tetrah. vol. 48, No. 12, 2223-2311, (1992); and Tetrah. vol. 48, No. 28, 6123-6194, (1993)]. This is particularly advantageous when synthesizing oligonucleotides which possess modified phosphate bridges.
The oligonucleotides are assembled using methods which are known to the person skilled in the art, such as the triester method, the H-phosphonate method or the phosphoramidite method [E. Sonveaux, (1986), Bioorganic Chemistry, 14, 274-325; S. L. Beaucage et al., (1992), Tetrahedron, 48, 2223-2311]. The nucleotide monomer structural components of the formula V, particularly preferably those of the formula V in which Exe2x80x2 is NR10R11 and Fxe2x80x2 is OR12, or Fxe2x80x2 is NR10NR11 and Exe2x80x2 is H, are preferably employed for introducing the 7-deazapurine derivatives.
The compounds of the formula V can be prepared, as structural components for the oligonucleotide solid phase synthesis, by proceeding from the corresponding 7-deazapurine nucleosides. Substituents can be introduced at the 7 position of the 7-deazapurine ring system using well-known methods. For example, the preparation of 7-deazapurine nucleosides which are substituted at the 7 position by halogen or methyl is described by Seela et al. [Helvetica Chimica Acta, (1994) 77, 897-903]. Alkenyl- or alkynyl-substituted 7-deazapurine derivatives of the formula V can be prepared by proceeding from the known 5-iodotubercidin (=7-I-7-deazaadenosine, see Seela et al., above), and coupling alkenyl or alkynyl groups onto the 7 position of the 7-deazapurine ring system by means of a cross-coupling reaction in the presence of tetrakis(triphenylphosphine)palladium(O). Electrophilic substituents (for example halogens) can be introduced into the 8 position of the 7-deazapurine ring system if nucleosides are employed as starting compounds which possess an electron-supplying substituent (for example an amino group) at the 2 position of the 7-deazapurine. If the 2-amino group is, for example, acetylated, the electrophilic substituent is then directed into the 7 position. Consequently, the present invention also relates to a process for the regioselective insertion of electrophilic substituents (for example halogens) into the 7 or 8 position of 7-deazanucleosides. The halogenated nucleosides can then be used as starting compounds for the insertion of other substituents, for example alkyl, alkenyl or alkynyl groups, by means of the above-described palladium-catalyzed cross-coupling reaction. Alkoxy derivatives or substituted amine derivatives can be introduced by nucleophilic substitution, and nitro groups can be introduced by electrophilic substitution.
After suitable protective groups for the amino groups of the 7-deazapurine bases and for the free 5xe2x80x2-hydroxyl group of the sugar have been introduced, the monomers are converted into the corresponding phosphonate or phosphoramidite derivatives. Suitable amino protective groups, for example in the form of a formamidine protective group ((dimethylamino)methylidene) or acyl protective groups (e.g. benzoyl or phenoxyacetyl), are inserted using well-known methods [L. J. McBride et al., (1983) Tetrahedron Lett., 24, 2953, G. S. Ti et al., (1982) J. Am. Chem. Soc., 104, 1316; H. Schaller et al. (1963), J. Am. Chem. Soc., 85, 3821], with it being advantageous, when the amino group is acylated, to use the Schaller peracylation method. An example of a suitable protective group for the free 5xe2x80x2-OH group of the sugar is 4,4xe2x80x2-dimethoxytrityl, whose insertion is likewise effected using known methods [C. B. Reese (1978), Tetrahedron, 34, 3143; D. Flockerzi et al., (1981), Liebigs Ann. Chem., 1568]. The monomers which have been protected in this way can be converted into the corresponding phosphonates in accordance with a protocol due to Froehler et al. [B. C. Froehler et al., (1986), Nucl. Acid Res., 14, 5399]. Cyanoethyl-phosphoramidite derivatives can, for example, be prepared by reacting the monomers with chloro-xcex2-cyanoethoxy-(N,N-diisopropylamino)phosphane in anhydrous dichloromethane [N. D. Sinha et al., (1984) Nucl. Acid Res., 12, 4539].
Compounds of the formula I whose oligonucleotide moiety is modified at the 3xe2x80x2 end and/or the 5xe2x80x2 end are synthesized, as regards these modifications, using the methods described in EP-A 0 552 766.
For use according to the invention, the oligonucleotides have a length of from 4 to 100, preferably of about 5-40, in particular of about 6-30, nucleotides. Otherwise, the above-described preference ranges, modifications and conjugations also apply in this case too.
The present invention relates to the use of oligonucleotides containing at least one substituted 7-deazapurine, preferably 7-deazaadenine or 7-deazaguanine, as a diagnostic reagent, for example for detecting the presence or absence of, or the quantity of, a specific double-stranded or single-stranded nucleic acid molecule in a biological sample. One or more of these oligonucleotides maybe directly or indirectly bound or absorbed onto a solid support, or provided as a solution in a solvent or diluent, optionally together with other conventional diagnostically relevant auxiliary reagents.
The invention furthermore relates to pharmaceutical compositions comprising one or more oligonucleotides of the formula I, together with a physiologically acceptable excipient and, where appropriate, suitable additives and/or conventional auxiliary substances.
In a quite general manner, the present invention extends to the use of oligonucleotides of the formula I in therapeutically effective amounts in improved therapeutic methods. In general, therapeutically effective oligonucleotide derivatives are understood to mean antisense oligonucleotides, triple helix-forming oligonucleotides, aptamers or ribozymes, in particular antisense oligonucleotides.
The pharmaceuticals of the present invention can, for example, be used to treat diseases which are caused by viruses, for example by HIV, HSV-1, HSV-2, influenza, VSV, hepatitis B or papilloma viruses.
Novel antisense oligonucleotide derivatives, that is antisense oligonucleotides in which at least one purine base is replaced by a substituted 7-deazapurine base, and which are effective against these targets, have, for example, the following base sequences:
a) against HIV, e.g.
5xe2x80x2-ACACCCAATTCTGAAAATGG-3xe2x80x2 (SEQ ID NO: 1) or (I)
5xe2x80x2-AGGTCCCTGTTCGGGCGCCA-3xe2x80x2 (SEQ ID NO: 2) or (II)
5xe2x80x2-GTCGACACCCAATTCTGAAAATGGATAAA-3xe2x80x2 (SEQ ID NO: 3) (III)
5xe2x80x2-GCTATGTCGACACCCAATTCTGAAA-3xe2x80x2 (SEQ ID NO: 4) or (IV)
5xe2x80x2-TCGTCGCTGTCTCCGCTTCTTCTTCCTGCCA-3xe2x80x2 (SEQ ID NO: 5) or (VI)
b) against HSV-1, e.g.
5xe2x80x2-GCGGGGCTCCATGGGGGTCG-3xe2x80x2 (SEQ ID NO: 6) (VII)
The pharmaceuticals of the present invention are also suitable, for example, for treating cancer. For example, oligonucleotide sequences can be used in this context which are directed against targets which are responsible for the occurrence of cancer or for cancer growth. Examples of such targets are:
1) nuclear oncoproteins such as, for example, c-myc, N-myc, c-myb, c-fos, c-fos/jun, PCNA and p120,
2) cytoplasmic/membrane-associated oncoproteins such as, for example, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos, c-src and c-abl,
3) cellular receptors, such as, for example, the EGF receptor, c-erbA, retinoid receptors, the protein kinase regulatory subunit and c-fms,
4) cytokines, growth factors, and extracellular matrix, such as, for example, CSF-1, IL-6, IL-1a, IL-1b, IL-2, IL-4, bFGF, myeloblastin and fibronectin.
Novel antisense oligonucleotides of the formula I which are effective against these targets have, for example, the following base sequences:
a) against c-Ha-ras, e.g.
5xe2x80x2-CAGCTGCAACCCAGC-3xe2x80x2 (SEQ ID NO: 7) (VIII)
c) c-myc, e.g.
5xe2x80x2-GGCTGCTGGAGCGGGGCACAC-3xe2x80x2 (SEQ ID NO: 8) (IX)
5xe2x80x2-AACGTTGAGGGGCAT-3xe2x80x2 (SEQ ID NO: 9) (X)
d) c-myb, e.g.
5xe2x80x2-GTGCCGGGGTCTTCGGGC-3xe2x80x2 (SEQ ID NO: 10) (XI)
e) c-fos, e.g.
5xe2x80x2-GGAGAACATCATGGTCGAAAG-3xe2x80x2 (SEQ ID NO: 11) (XII)
5xe2x80x2-CCCGAGAACATCATGGTCGAAG-3xe2x80x2 (SEQ ID NO: 12) (XIII)
5xe2x80x2-GGGGAAAGCCCGGCAAGGGG-3xe2x80x2 (SEQ ID NO: 13) (XIV)
f) p120, e.g.
5xe2x80x2-CACCCGCCTTGGCCTCCCAC-3xe2x80x2 (SEQ ID NO: 14) (XV)
g) EGF receptor, e.g.
5xe2x80x2-GGGACTCCGGCGCAGCGC-3xe2x80x2 (SEQ ID NO: 15) (XVI)
5xe2x80x2-GGCAAACTTTCTTTTCCTCC-3xe2x80x2 (SEQ ID NO: 16) (XVII)
h) p53 tumor suppressor, e.g.
5xe2x80x2-GGGAAGGAGGAGGATGAGG-3xe2x80x2 (SEQ ID NO: 17) (XVIII)
5xe2x80x2-GGCAGTCATCCAGCTTCGGAG-3xe2x80x2 (SEQ ID NO: 18) (XIX)
The pharmaceuticals of the present invention are furthermore suitable, for example, for treating diseases which are affected by integrins or cell-cell adhesion receptors, for example by VLA-4, VLA-2, ICAM, VCAM or ELAM.
Novel antisense oligonucleotide derivatives which are effective against these targets have, for example, the following base sequences:
a) VLA-4, e.g.
5xe2x80x2-GCAGTAAGCATCCATATC-3xe2x80x2 (SEQ ID NO: 19) or (XX)
b) ICAM, e.g.
5xe2x80x2-CCCCCACCACTTCCCCTCTC-3xe2x80x2 (SEQ ID NO: 20) (XXI)
5xe2x80x2-CTCCCCCACCACTTCCCCTC-3xe2x80x2 (SEQ ID NO: 21) (XXII)
5xe2x80x2-GCTGGGAGCCATAGCGAGG-3xe2x80x2 (SEQ ID NO: 22) (XXIII)
c) ELAM-1, e.g.
5xe2x80x2-ACTGCTGCCTCTTGTCTCAGG-3xe2x80x2 (SEQ ID NO: 23) (XXIV)
5xe2x80x2-CAATCAATGACTTCAAGAGTTC-3xe2x80x2 (SEQ ID NO: 24) (XXV)
The pharmaceuticals of the present invention are also suitable, for example, for preventing restenosis. For example, oligonucleotide sequences can be used in this context which are directed against targets which are responsible for proliferation or migration. Examples of these targets are:
1) Nuclear transactivator proteins and cyclins, such as, for example, c-myc, c-myb, c-fos, c-fos/jun, cyclins and cdc2 kinase
2) Mitogens or growth factors, such as, for example, PDGF, bFGF, EGF, HB-EGF and TGF-xcex2.
3) Cellular receptors such as, for example, bFGF receptor, EGF receptor and PDGF receptor.
Novel oligonucleotides of the formula I which are effective against these targets have, for example, the following base sequences:
a) c-myb
5xe2x80x2-GTGTCGGGGTCTCCGGGC-3xe2x80x2 (SEQ ID NO: 25) (XXVI)
b) c-myc
5xe2x80x2-CACGTTGAGGGGCAT-3xe2x80x2 (SEQ ID NO: 26) (XXVII)
c) cdc2 kinase
5xe2x80x2-GTCTTCCATAGTTACTCA-3xe2x80x2 (SEQ ID NO: 27) (XXVIII)
d) PCNA (proliferating cell nuclear antigen of rat)
5xe2x80x2-GATCAGGCGTGCCTCAAA-3xe2x80x2 (SEQ ID NO: 28) (XXIX)
The pharmaceuticals can be used, for example, in the form of pharmaceutical preparations which can be administered orally, for example in the form of tablets, coated tablets, hard or soft gelatin capsules, solutions, emulsions or suspensions. The inclusion of the pharmaceuticals in liposomes, which, where appropriate, contain additional components such as proteins, likewise represents a suitable administration form. They can also be administered rectally, for example in the form of suppositories, or parenterally, for example in the form of injection solutions. For the production of pharmaceutical preparations, these compounds can be processed in therapeutically inert, organic and inorganic excipients. Examples of such excipients for tablets, coated tablets and hard gelatin capsules are lactose, corn starch, or derivatives thereof, tallow and stearic acid, or salts thereof. Suitable excipients for preparing solutions are water, polyols, sucrose, invert sugar and glucose. Suitable excipients for injection solutions are water, alcohols, polyols, glycerol and vegetable oils. Suitable excipients for suppositories are vegetable and hardened oils, waxes, fats and semiliquid polyols. The pharmaceutical preparations can also contain preservatives, solvents, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for altering the osmotic pressure, buffers, coating agents, antioxidants and other therapeutical active compounds, where appropriate.
Preferred forms of administration are topical administrations, local administrations, such as, for example, using a catheter, or else injections. For injection, the antisense oligonucleotide derivatives are formulated in a liquid solution, preferably in a physiologically acceptable buffer, such as, e.g., Hank""s solution or Ringer""s solution. However, the antisense oligonucleotides can also be formulated in solid form and dissolved or suspended prior to use. The doses which are preferred for systemic administration amount to from about 0.01 mg/kg to about 50 mg/kg of body weight and per day.
In a quite general manner, the invention extends to the use of compounds of the formula I as DNA probes or primers in DNA diagnostics and, in a general manner, as aids in molecular biology , as noted earlier.
Individual DNA molecules can be visualized electron microscopically, for example in a scanning-tunneling microscope. While pyrimidine bases can be differentiated electronmicroscopically due to the methyl group at the 5 position, this is not possible in the case of the purine bases adenine and guanine. It is not possible, therefore, to decode the base sequences of nucleic acid molecules electronmicroscopically in a straightforward manner. However, if the nucleic acid molecule to be investigated now contain substituted 7-deazaguanine derivatives, for example, in place of the unmodified guanine bases, the substituted 7-deazaguanine bases can be distinguished in the electron microscope from unsubstituted adenine bases (and, conversely, guanine bases can be distinguished from substituted 7-deazaadenine bases). Consequently, the base sequences of nucleic acids which contain 7-substituted 7-deazapurine bases can be decoded by electron microscopy.